In nonexcitable cells, the depletion of internal Ca2+ stores leads to the activation of store-operated channels (SOCs) which play an important role in physiological processes such as refilling of Ca2+ stores, regulation of secretion, regulation of gene transcription, control of cell cycle and proliferation, and regulation of apoptosis. Until recently, what little was known about the signaling mechanism for coupling depletion of Ca2+ stores to activation of SOCs was based on a number of pharmacological studies which implicated a variety of potential regulatory pathways. For example, based on studies with a series of tyrosine kinase and tyrosine phosphatase inhibitors, a role for tyrosine kinases in the regulation of SOCs was proposed. During the previous grant period, a molecular approach provided evidence for the involvement of c-src and focal adhesion kinase (FAK) in the regulation of SOCs. To our knowledge, this makes the tyrosine kinase pathway the first to have both pharmacological and molecular data supporting its involvement in the signaling mechanism for activation of SOCs. In this proposal, experiments are described to elucidate the events downstream of c-src and FAK in the activation of SOCs. This will extend the studies which demonstrate that store-operated Ca2+ entry (SOCE) is dramatically reduced in fibroblasts from transgenic src_ mice, but can be restored by transfecting wild type c-src into these cells. Experiments are proposed specifically to 1) express a series of src mutants to determine which domains of c-src are necessary to activate SOCE. In addition, mutants of FAK, Grb2 and Shc, which are known to interfere with signaling downstream from the src/FAK complex in the integrin signaling pathway, will be expressed to determine whether they block activation of SOCE. The primary hypothesis is that the tyrosine kinase activity of c-src is both necessary and sufficient to activate SOCE. 2) Determine which proteins are involved in SOCE in HEK-293 cells as a means of identifying potential targets of tyrosine kinase activity of c-src and FAK. While there is strong evidence that trp is a SOC in Drosophila, there is still uncertainty concerning which of the 7 mammalian trp homologs identified to date are SOCs. In the last grant period 4 trp homologs were identified as being expressed in HEK-293 cells and antisense constructs specific for each were made. The expression of individual trp homologs will be blocked and the effect on SOCE assessed by fura-2 and electrophysiological approaches. 3) Determine whether the protein responsible for SOCE in HEK-293 cells is directly tyrosine phosphorylated when SOCE is activated. If this proves negative, he will investigate the tyrosine phosphorylation of accessory proteins which directly interact with trp proteins (e.g. calmodulin and the human homolog of InaD, a regulatory protein for Drosophilia trp). The successful outcome of these experiments would create a significant breakthrough in our understanding of how SOCs are regulated.